A method for high-yield production of 2-hydroxyphenazine by pseudomonas

Abstract

This invention relates to the field of genetic engineering technology, and provides a method for high-yield production of 2-hydroxyphenazine using Pseudomonas aeruginosa, using strain Qlu-1ΔHΔPΔO as the starting strain, and knocking out pykA Genes were used to obtain strain Qlu-1ΔHΔPΔO-1; pykA The gene sequence is shown in SEQ ID NO. 1. This invention uses *Pseudomonas aeruginosa* Qlu-1ΔHΔPΔO from ZL 202210461035.X as the starting strain, and employs a scarless knockout method... pykA Genes were used to obtain the engineered strain Qlu-1ΔHΔPΔO-1. After fermentation and yield testing, it was found that the 2-hydroxyphenazine yield of Qlu-1ΔHΔPΔO-1 was increased to 321.2 mg / L, thus achieving an increase in the yield of 2-hydroxyphenazine in the strain.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a method for high-yield 2-hydroxyphenazine using Pseudomonas. Background Technology

[0002] The information disclosed in the background section of this invention is intended only to enhance the understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Since modern times, chemically synthesized pesticides have been crucial for high and stable crop yields. However, the extensive use of these non-degradable pesticides easily leads to pesticide residues in grains, fruits, and vegetables, causing environmental pollution and contradicting the trend of sustainable development in today's society. Biopesticides, on the other hand, are a class of bioactive substances derived from or produced by organisms. Compared to the widely used chemically synthesized pesticides, biopesticides have significant advantages such as low toxicity, environmental friendliness, easy degradation, and low likelihood of developing resistance, attracting increasing attention and gradually being applied in biological control practices. Among them, phenazine compounds produced by Pseudomonas and Streptomyces are representative of a class of highly efficient biopesticides.

[0004] 2-Hydroxyphenazine is a nitrogen-containing heterocyclic bioactive substance that has a good control effect on fungal diseases of various crops. Previous experiments have shown that 2-hydroxyphenazine can be used to control wheat scab, wheat root rot, rice sheath blight, rice blight, pepper blight, and melon vine blight. In some diseases, its control efficacy is better than that of similar herbicides such as shenqinmycin (whose active ingredient is 1% phenazine-1-carboxylic acid). Therefore, it is a potential new type of biological pesticide.

[0005] Currently, although there are chemical methods for producing 2-hydroxyphenazine, the conditions are harsh and the synthesis efficiency is low. *Pseudomonas aeruginosa* (… Pseudomonas chlororaphis Qlu-1 is a *Pseudomonas aeruginosa* strain selected from plant rhizosphere soil, capable of synthesizing phenazine compounds. phzABCDEFG Gene clusters can produce phenazine-1-carboxylic acid and 2-hydroxyphenazine, etc.

[0006] To increase the yield of 2-hydroxyphenazine, researchers have genetically modified wild-type strains. The applicant's prior patent (ZL 202210461035.X) discloses a high-yielding strain, Qlu-1ΔHΔPΔO, which, by knocking out some negative regulatory genes, significantly increases the yield of 2-hydroxyphenazine compared to the wild type. However, the yield of existing engineered strains is still insufficient to meet the cost and efficiency requirements of industrial production. How to further overcome the metabolic limitations of the strains and improve the synthesis efficiency of the target product remains a pressing technical problem to be solved in this field. Summary of the Invention

[0007] In view of this, the present invention provides a method for high-yield production of 2-hydroxyphenazine using Pseudomonas aeruginosa. The present invention uses Qlu-1ΔHΔPΔO(Qlu Using a derivative strain of strain 1 as the starting strain, the 2-hydroxyphenazine yield of the strain was increased to 321.2 mg / L through genetic engineering.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a genetically engineered bacterium that produces high levels of 2-hydroxyphenazine from Pseudomonas, using strain Qlu-1ΔHΔPΔO as the starting strain, and knocking out... pykA Genes were used to obtain strain Qlu-1ΔHΔPΔO-1; pykA The gene sequence is shown in SEQ ID NO. 1.

[0009] The strain Qlu-1ΔHΔPΔO of the present invention is Qlu The derivative strain of 1 was disclosed in ZL 202210461035.X.

[0010] pykA Gene knockout primers (primers for obtaining the pykA-UD fusion fragment) include: pykA-F1 / pykA-R1, pykA-F2 / pykA-R2.

[0011] The sequence of pykA-F1 is shown in SEQ ID NO. 3.

[0012] The sequence of pykA-R1 is shown in SEQ ID NO. 4.

[0013] The sequence of pykA-F2 is shown in SEQ ID NO. 5.

[0014] The sequence of pykA-R2 is shown in SEQ ID NO. 6.

[0015] A second aspect of the present invention provides a method for preparing a genetically engineered bacterium that produces high levels of 2-hydroxyphenazine from Pseudomonas, using strain Qlu-1ΔHΔPΔO as the starting strain, and knocking out... pykA Genes were extracted to obtain strain Qlu-1ΔHΔPΔO-1.

[0016] In some embodiments, the preparation method specifically includes the following steps: PCR amplification of *Pseudomonas aeruginosa* (… Pseudomonas chlororaphis Qlu-1 pykA The upstream and downstream sequence fragments of the gene, pykA-UD, were ligated into plasmid pk18mobsacB to construct... pykAThe gene knockout plasmid pK18-pykA-UD was introduced into Escherichia coli via heat shock transformation and co-cultured with Pseudomonas aeruginosa Qlu-1ΔHΔPΔO to screen for knockout. pykA The strain is strain Qlu-1ΔHΔPΔO-1.

[0017] Among them, Pseudomonas aeruginosa ( Pseudomonas chlororaphis Qlu-1 is the *Pseudomonas aeruginosa* described in patent ZL202210461035.X. Pseudomonas chlororaphis Qlu-1.

[0018] In some embodiments, the pykA The gene sequence is shown in SEQ ID NO. 1, and the pykA-UD sequence is shown in SEQ ID NO: 2.

[0019] In some embodiments, the preparation method specifically includes the following steps: PCR amplification of *Pseudomonas aeruginosa* (… Pseudomonas chlororaphis Qlu-1 pykA The upstream and downstream sequence fragments pykA-UD were amplified using the Qlu-1ΔHΔPΔO genome as a template and pykA-F1 / pykA-R1 and pykA-F2 / pykA-R2 as primers, respectively. pykA The upstream sequence of the gene is pykA-U. pykA The downstream sequence pykA-D was obtained. Using pykA-U and pykA-D as templates and pykA-F1 / pykA-R2 as primers, the pykA-U and pykA-D fragments were ligated by fusion PCR to obtain the fusion fragment pykA-UD. pK18-pykA-UD was then introduced into *E. coli* via heat shock transformation and co-cultured with *Pseudomonas aeruginosa* Qlu-1ΔHΔPΔO. Knockout was screened and verified by PCR. pykA Strain Qlu-1ΔHΔPΔO-1.

[0020] The sequence of pykA-F1 is shown in SEQ ID NO. 3.

[0021] The sequence of pykA-R1 is shown in SEQ ID NO. 4.

[0022] The sequence of pykA-F2 is shown in SEQ ID NO. 5.

[0023] The sequence of pykA-R2 is shown in SEQ ID NO. 6.

[0024] In a third aspect, the present invention provides a strain Qlu-1ΔHΔPΔO-1 prepared by the above method.

[0025] A fourth aspect of the present invention provides a method for high-yield production of 2-hydroxyphenazine using Pseudomonas, comprising: The above-mentioned strain Qlu-1ΔHΔPΔO-1 was inoculated into a fermentation medium to produce 2-hydroxyphenazine.

[0026] The fermentation medium is selected from KB medium.

[0027] Compared with the prior art, the present invention has achieved the following beneficial effects: (1) This invention uses the publicly disclosed high-yield strain Qlu-1ΔHΔPΔO as the starting strain, and specifically knocks out the gene in its genome through genetic engineering. pykA Gene. pykA The gene encodes pyruvate kinase, a key rate-limiting enzyme in the glycolysis pathway that catalyzes the conversion of phosphoenolpyruvate to pyruvate. Knocking out this gene may alter the strain's carbon metabolic flux, allowing more carbon skeleton and reducing power (NADPH) to flow to the synthesis pathway of phenazine compounds while ensuring the bacteria's basic primary metabolic needs are met, thus removing the original metabolic bottleneck. Experimental results show that the engineered strain Qlu-1ΔHΔPΔO-1, obtained through the above modification, achieved a 2-hydroxyphenazine yield of 321.2 mg / L after 72 hours of fermentation in KB medium. Compared with the starting strain, the yield was significantly increased, directly reducing the unit fermentation cost of 2-hydroxyphenazine and laying a solid foundation for its industrial production. Attached Figure Description

[0028] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments and descriptions of the invention are used to explain the invention and do not constitute an undue limitation thereof. Obviously, those skilled in the art can obtain other drawings based on these drawings without any inventive effort.

[0029] Figure 1 Electrophoresis diagram of the mutant plasmid pK18-pykA-UD in Example 1; where, (A) pykA Amplification of upstream and downstream homologous arm fragments: 1, pykA Upstream homologous arm amplification; 2, pykA Upstream homologous arm amplification; 3, DNA Ladder DL5000; 4, pykA Downstream homologous arm fragment amplification; 5, pykA Downstream homologous arm fragment amplification; (B) pykA Amplification of upstream and downstream homologous arm fusion fragments: 1, pykA Upstream and downstream homologous arm fusion fragments; 2, pykA 3. Upstream and downstream homologous arm fusion fragments; 4. DNA LadderDL5000; pykA Upstream and downstream homologous arm fusion fragments; 5, pykA Upstream and downstream homologous arm fusion fragment.

[0030] Figure 2 In Example 1 pykA PCR validation diagram of the knockout strain; where, external primer detection: 1, blank control; 2, DNALadder DL5000; 3, with pykA The gene knockout strain Qlu-1ΔHΔPΔO-1 genome was used as a template for amplification of the fragment; 4. The Qlu-1ΔHΔPΔO genome was used as a template for amplification of the fragment; Internal primer detection: 1. DNA Ladder DL5000; 2. The Qlu-1ΔHΔPΔO genome was used as a template for amplification of the fragment; 3. The Qlu-1ΔHΔPΔO genome was used as a template for amplification of the fragment; pykA The genome of the gene knockout strain Qlu-1ΔHΔPΔO-1 was used as a template for amplification; 4, blank control.

[0031] Figure 3 The yield of 2-hydroxyphenazine after 72 hours of fermentation by different strains. Detailed Implementation

[0032] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0033] Example 1 (1) Inoculate Pseudomonas green needle Qlu-1 into King'B medium, and culture overnight at 30°C with a shaker at 180 rpm. Extract the genome of Pseudomonas green needle Qlu-1 using a prokaryotic genome extraction kit and store at -20°C for later use.

[0034] (2) Searching for genome data of the sequenced Pseudomonas green needle Qlu-1 pykA Gene and upstream / downstream sequences: Using the genome of *Pseudomonas aeruginosa* Qlu-1ΔHΔPΔO as a template, and using pykA-F1 / pykA-R1 and pykA-F2 / pykA-R2 as primers, the upstream sequence pykA-U of pykA was amplified. pykA The downstream sequence pykA-D was obtained by using pykA-U and pykA-D as templates and pykA-F1 / pykA-R2 as primers to ligate the pykA-U and pykA-D fragments using fusion PCR to obtain the fusion fragment pykA-UD.

[0035] (3) The fusion fragment pykA-UD was ligated with the knockout plasmid pk18mobsacB to construct the recombinant plasmid pk18-pykA-UD. Figure 1 ).

[0036] (4) The recombinant plasmid pk18-pykA-UD was introduced into Escherichia coli S17-1 (λpir) by heat shock transformation.

[0037] (5) Escherichia coli S17-1(λpir) and Pseudomonas aeruginosa Qlu-1ΔHΔPΔO were hybridized to each parent and the recombinant plasmid pk18-pykA-UD was introduced into Pseudomonas aeruginosa Qlu-1ΔHΔPΔO.

[0038] (6) The mutant strain Qlu-1ΔHΔPΔO-1 was obtained by sucrose plate screening and photocopy screening.

[0039] (7) Verify the Qlu-1ΔHΔPΔO using PCR verification method. pykA Knockout strain Qlu-1ΔHΔPΔO-1. Figure 2 ) Strains Qlu-1ΔHΔPΔO-1, Qlu-1, and Qlu-1ΔHΔPΔO were inoculated into KB medium and fermented at 30°C. 2-hydroxyphenazine was extracted from the fermentation broth. HPLC analysis revealed that strain Qlu-1ΔHΔPΔO-1 produced 321.2 mg / L of 2-hydroxyphenazine after 72 hours. Figure 3 As shown.

[0040] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A genetically engineered bacterium that utilizes Pseudomonas to produce high levels of 2-hydroxyphenazine, characterized in that, Using strain Qlu-1ΔHΔPΔO as the starting strain, knockout pykA Genes were used to obtain strain Qlu-1ΔHΔPΔO-1; pykA The gene sequence is shown in SEQ ID NO.

1.

2. A method for preparing a genetically engineered bacterium that produces high levels of 2-hydroxyphenazine from Pseudomonas, characterized in that, Using strain Qlu-1ΔHΔPΔO as the starting strain, knockout pykA Genes were extracted to obtain strain Qlu-1ΔHΔPΔO-1.

3. The method for preparing a genetically engineered bacterium that produces high levels of 2-hydroxyphenazine from Pseudomonas as described in claim 2, characterized in that, The preparation method specifically includes the following steps: PCR amplification of *Pseudomonas aeruginosa* (… Pseudomonas chlororaphis Qlu-1 pykA Gene and upstream and downstream sequence fragments pykA-UD, pykA-UD fragment ligated into plasmid pk18mobsacB to construct pykA The gene knockout plasmid pK18-pykA-UD was introduced into Escherichia coli via heat shock transformation and co-cultured with Pseudomonas aeruginosa Qlu-1ΔHΔPΔO to screen for knockout. pykA The strain is strain Qlu-1ΔHΔPΔO-1.

4. The method for preparing a genetically engineered bacterium that produces high levels of 2-hydroxyphenazine from Pseudomonas as described in claim 3, characterized in that, The pykA The gene sequence is shown in SEQ ID NO. 1, and the pykA-UD sequence is shown in SEQ ID NO:

2.

5. The strain Qlu-1ΔHΔPΔO-1 prepared by the method according to any one of claims 2-4.

6. A method for high-yield production of 2-hydroxyphenazine using Pseudomonas, characterized in that, include: The strain Qlu-1ΔHΔPΔO-1 according to any one of claims 1 and 5 is inoculated into a fermentation medium to produce 2-hydroxyphenazine.

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